The fact that high levels of retinyl esters have been measured in normal epithelia throughout the body suggests that these retinyl esters play some role in the function, growth, or differentiation of these normal epithelial cells. Furthermore, retinoids are known to control normal differentiation and can convert preneoplastic epithelial cells to a more normal phenotype. Nevertheless, the functions of these retinyl esters are not well understood. We have recently shown that whereas cultured normal human epithelial cell strains from the oral cavity and skin rapidly esterify retinol, human squamous cell carcinoma cell lines from the oral cavity and skin exhibit almost no retinol esterification. Therefore, we plan to analyze the molecular actions of these retinyl esters and to determine how the synthesis of these retinyl esters is regulated. To accomplish this, we propose to clone and biochemically characterize the enzyme(s), ARAT (acyl-CoA: retinol acyltransferase) and LRAT (lecithin:retinol acyltransferase), involved in the synthesis of retinyl esters from retinol in normal epithelial cells. We will determine why ARAT levels are low in the squamous cell carcinomas (SCCs) as compared to the normal epithelial cells. We also propose to establish to what extent this defect in retinol esterification is associated with aspects of the tumorgenic phenotype. This will be accomplished first by stably transfecting the ARAT sense cDNA, under the control of a tetracycline regulated promoter, into the human SCC lines and measuring cell growth, invasiveness, and differentiation associated gene expression in cells cultured tet, or by blocking the expression of ARAT in normal cells. We also propose to test whether this defect in the conversion of retinol to retinyl esters is observed in an animal model of carcinogenesis which recapitulates most of the development of human SCC of the oral cavity. In addition, we will generate mice in which the ARAT gene is ectopically expressed in the oral cavity of transgenic animals to determine whether this ectopic expression will reduce tumor development after treatment of the oral cavities of the mice with 4-nitroquinoline-1-oxide. These proposed studies should determine the importance of retinyl esters in normal epithelial cells of the oral cavity and skin and the relevance of the loss of these retinyl esters in establishing the transformed state. Moreover, if a high level of retinol esterification is shown to be critical for normal cell functioning, then it may be possible to develop drugs which stimulate retinol esterification in tumor cells as a therapy for human squamous cell carcinomas of the oral cavity and skin.